Comparison between two protocols of cryopreservation (one or two steps), thawing (fast and slow) and proposal of a functional canine sperm analysis test

Abstract

The present study aims to compare two protocols of seminal cryopreservation, two temperatures of thawing canine semen, as well as standardization and evaluation of the efficiency of the mobile sperm binding to egg yolk membrane. Five male dogs will be selected, on reproductive age and free from any disease. The stud dog will have their semen collected individually and separated in two groups: Group 1 - the semen will be cryopreservated in one step (adding the extender with glycerol at 37ºC), and Group 2 - cryopreservation in two steps (adding glycerol after refrigeration of the pre-extended semen). On the thawing process, the different groups will be tested for two protocols: Thawing A - 37ºC for 30 seconds and Thawing B - 70ºC for 8 seconds. The evaluation of the pre-freezing and post-thawing samples will be: subjective sperm motility, sperm velocity, computer analysis of the motility (CASA), oxidative stress (induced TBARS), mitochondrial activity (DAB), plasmatic membrane permeability (Eosin/Nigrosin), sperm morphology (saline formaline), acrossomal integrity (POPE) and test of the mobile sperm binding to egg yolk membrane. The results will be compared to verify which cryopreservation technique guarantees higher survival of the sperm and if the test of the mobile sperm binding to egg yolk membrane can be used as a functional method of canine semen analyses. (AU)